Pseudomonas putida is a bacterium which can grow on D-camphor as ts source of carbon and energy. In the presence of camphor this organism contains a soluble cytochrome P-450-type of monooxygenase system. The membrane-bound electron transport system which occurs in this organism is unusual in that the terminal oxygen reducing enzyme is not a heme A type of cytochrome. The ultimate goal of this research program is to understand the mechanism of oxygen reduction by these two systems and their interaction. RESEARCH GOALS FOR THE COMING YEAR: A) Studies of the sequence of electron carriers in the membrane-bound electron transport system of P. putida will be carried out using the stopped-flow spectrophotometer which was developed for the study of turbid suspensions. The interaction of the cytochrome P-450 system and the membrane bound electron transport system will also be studied using whole cells and sensitive spectrophotometric and fluorimetric techniques to monitor the states of these enzymes during the steady state of oxygen consumption and camphor metabolism. B) The cytochrome P-450-dependent oxidation of camphor requires an electron transport chain consisting of a flavoprotein and an iron-sulfur protein. The proposed research will investigate the interaction of these proteins and cytochrome P-450 during electron transfer using a freeze-quench technique to prepare samples for EPR spectroscopy. Using this technique we hope to investigate the process of electron transfer to cytochrome P-450 and the "effector" roles of the electron transfer proteins in modifying and controlling this reaction. C) The crystals of the camphor complex of cytochrome P-450cam will be employed in a study of the structure of the active site of the enzyme. Preliminary results indicate that the crystals are sufficiently stable to form various derivatives of the heme iron.